Glycine 1.50 g Na2SO4 3.00 g KH2PO4 0.20 g MgCl2 x 6 H2O 0.40 g NaCl 1.20 g CaCl2 x 2 H2O 0.15 g KCl 0.40 g Yeast extract 0.20 g Trace element solution SL-10 1.00 ml Selenite-tungstate solution 1.00 ml Sodium resazurin (0.1% w/v) 0.50 ml NaHCO3 2.00 g Wolin's vitamin solution (10x) 1.00 ml Na2S x 9 H2O 0.30 g Distilled water 1000.00 ml Dissolve ingredients (except bicarbonate, vitamins and sulfide), adjust pH to 6.5, then sparge with 100% N2 gas for 30 - 45 min to make it anoxic and dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials. After autoclaving complete the medium by adding vitamins and sulfide from sterile anoxic stock solutions prepared under 100% N2 gas and bicarbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Vitamins are sterilized by filtration. Adjust pH of the complete medium to 7.4 - 7.5, if necessary. Trace element solution SL-10 (from medium 320) HCl (25%) 10.00 ml FeCl2 x 4 H2O 1.50 g ZnCl2 70.00 mg MnCl2 x 4 H2O 100.00 mg H3BO3 6.00 mg CoCl2 x 6 H2O 190.00 mg CuCl2 x 2 H2O 2.00 mg NiCl2 x 6 H2O 24.00 mg Na2MoO4 x 2 H2O 36.00 mg Distilled water 990.00 ml First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml. Selenite-tungstate solution (from medium 385) NaOH 0.50 g Na2SeO3 x 5 H2O 3.00 mg Na2WO4 x 2 H2O 4.00 mg Distilled water 1000.00 ml W'olins vitamin solution (10x) (from medium 120) Biotin 20.00 mg Folic acid 20.00 mg Pyridoxine hydrochloride 100.00 mg Thiamine HCl 50.00 mg Riboflavin 50.00 mg Nicotinic acid 50.00 mg Calcium D-(+)-pantothenate 50.00 mg Vitamin B12 1.00 mg p-Aminobenzoic acid 50.00 mg (DL)-alpha-Lipoic acid 50.00 mg Distilled water 1000.00 ml
